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目的研究整合素连接激酶(ILK)在细胞中的功能,构建ILK基因sh RNA慢病毒载体,并对其在舌鳞癌细胞株Tca-8113中沉默效果进行鉴定。方法针对ILK基因有效靶序列的3个位点,设计合成3对oligo DNA,退火形成双链DNA,与线性化p ENTR/U6载体连接产生sh ILK-LV慢病毒载体,筛选出阳性克隆测序鉴定,用sh ILK-LV载体、包装质粒Packaging Mix共转染293T包装细胞,包装产生慢病毒,以293T细胞中绿色荧光蛋白(GFP)的表达数目测定病毒滴度并确定恰当的MOI值。获得重组慢病毒后感染人舌鳞癌细胞株Tca-8113,用q PCR及Western blot检测ILK在Tca-8113细胞中的表达。结果测序证实成功构建了3个ILK-sh RNA慢病毒载体,分别转染Tca-8113细胞后用sybr法检测出sh RNA-ILK-370组ILK的表达明显下降,ΔΔCt值为0.268。用sh RNA-ILK-370慢病毒载体包装病毒,测定病毒滴度,并得出当MOI=30-60时,细胞阳性比率最高。用病毒感染Tca-8113细胞后再用抗生素筛选稳转株,后用q PCR检测干扰效率达90.5%,Western blot检测ILK表达明显被抑制。结论成功构建sh ILK-LV慢病毒载体并建立了稳定的Tca-8113-sh ILK细胞模型,为研究ILK在舌鳞癌信号转导通路中的作用提供了坚实基础。
Objective To study the function of ILK in cells and to construct lentiviral vector of ILK gene sh RNA and to identify its silencing effect in tongue squamous cell carcinoma cell line Tca-8113. Methods Three pairs of oligo DNAs were designed and synthesized based on the three active sites of ILK gene. The double-stranded DNAs were annealed to form sh ILK-LV lentiviral vector with linearized p ENTR / U6 vector. The positive clones were identified by sequencing The 293T packaging cells were cotransfected with sh ILK-LV vector and packaging plasmid Packaging Mix. The lentivirus was packaged and the virus titer was determined by the number of green fluorescent protein (GFP) in 293T cells and the appropriate MOI value was determined. Tca-8113 cells were infected with recombinant lentivirus and the expression of ILK in Tca-8113 cells was detected by q PCR and Western blot. Results Sequencing confirmed that three ILK-sh RNA lentivirus vectors were successfully constructed. The expression of ILK in sh RNA-ILK-370 group was significantly decreased after transfected with Tca-8113 cells using sybr method. The ΔΔCt value was 0.268. The virus was packaged with the sh RNA-ILK-370 lentiviral vector and the virus titer was determined, and the highest cell-positive ratio was found when MOI = 30-60. Tca-8113 cells were infected with virus and then the stable strains were screened by antibiotics. The interference efficiency was 90.5% by q PCR. The expression of ILK was significantly inhibited by Western blot. Conclusion The sh ILK-LV lentiviral vector was successfully constructed and a stable Tca-8113-sh ILK cell model was established, which provided a solid foundation for studying the role of ILK in the signal transduction pathway of tongue squamous cell carcinoma.